Method for detecting nucleic acid amplification using self-quenching fluorescence probe
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First Claim
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1. A method for monitoring nucleic acid amplification comprising:
- performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5'"'"'-3'"'"' nuclease activity, a primer capable of hybridizing to said target polynucleotide, and an oligonucleotide probe capable of hybridizing to said target polynucleotide, 3'"'"' relative to said primer,said oligonucleotide probe having a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule,said oligonucleotide probe existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one conformation when hybridized to said target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule being greater than the fluorescence intensity of said quencher molecule when said probe is hybridized to said target;
polynucleotide,said nucleic acid polymerase digesting said oligonucleotide probe during amplification to separate said reporter molecule from said quencher molecule; and
monitoring the fluorescence of said reporter molecule, the generation of fluorescence corresponding to the occurrence of nucleic acid amplification.
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Abstract
A method is provided for monitoring the progress of nucleic acid amplifications that rely on a nucleic acid polymerase having 5'"'"'→3'"'"' exonuclease activity. An important feature of the method is providing an oligonucleotide probe having a reporter molecule and a quencher molecule at either end such that the quencher molecule substantially quenches any fluorescence from the reporter whenever the oligonucleotide probe is in a single stranded state and such that the reporter is substantially unquenched whenever the oligonucleotide probe is in a double stranded state hybridized to a target polynucleotide.
952 Citations
24 Claims
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1. A method for monitoring nucleic acid amplification comprising:
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performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5'"'"'-3'"'"' nuclease activity, a primer capable of hybridizing to said target polynucleotide, and an oligonucleotide probe capable of hybridizing to said target polynucleotide, 3'"'"' relative to said primer, said oligonucleotide probe having a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one conformation when hybridized to said target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule being greater than the fluorescence intensity of said quencher molecule when said probe is hybridized to said target;
polynucleotide,said nucleic acid polymerase digesting said oligonucleotide probe during amplification to separate said reporter molecule from said quencher molecule; and monitoring the fluorescence of said reporter molecule, the generation of fluorescence corresponding to the occurrence of nucleic acid amplification. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 23)
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14. A method for monitoring nucleic acid amplification comprising:
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performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5'"'"'-3'"'"' nuclease activity, a primer capable of hybridizing to said target polynucleotide, and an oligonucleotide probe capable of hybridizing to said target polynucleotide 3'"'"' relative to said primer, said oligonucleotide probe having a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one conformation when hybridized to said target polynucleotide where the fluorescence of said reporter molecule is unquenched the fluorescence intensity of said oligonucleotide probe hybridized to said target polynucleotide being at least about a factor of 6 greater than the fluorescence intensity of said oligonucleotide probe when not hybridized to said target polynucleotide, said nucleic acid polymerase digesting said oligonucleotide probe during amplification to separate said reporter molecule from said quencher molecule; and monitoring the fluorescence of said reporter molecule, the generation of fluorescence corresponding to the occurrence of nucleic acid amplification. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22)
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24. A method for monitoring nucleic acid amplification comprising:
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performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5'"'"'-3'"'"' nuclease activity, a primer capable of hybridizing to said target polynucleotide and an oligonucleotide probe capable of hybridizing to said target polynucleotide 3'"'"' relative to said primer, said oligonucleotide probe having a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one conformation when hybridized to said target polynucleotide where the fluorescence of said reporter molecule is unquenched, the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said probe is hybridized to said target polynucleotide being at least about a factor of 6 greater than the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said probe is single-stranded, said nucleic acid polymerase digesting said oligonucleotide probe during amplification to separate said reporter molecule from said quencher molecule; and monitoring the fluorescence of said reporter molecule, the generation of fluorescence corresponding to the occurrence of nucleic acid amplification.
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Litigation Data
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Current AssigneeApplied Biosystems LLC (Thermo Fisher Scientific Incorporated)
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Original AssigneeApplied Biosystems Division Perkin-Elmer Corporation
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InventorsMarmaro, Jeffrey, Livak, Kenneth J., Flood, Susan J. A.
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Primary Examiner(s)FREDMAN, JEFFREY NORMAN
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Assistant Examiner(s)Rees, Dianne
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Application NumberUS08/340,558Time in Patent Office615 DaysField of Search435/6, 435/91.2, 435/5, 536/24.3-.33, 536/26.6US Class Current435/6.16CPC Class CodesC12Q 1/6818 involving interaction of tw...C12Q 1/6834 Enzymatic or biochemical co...C12Q 1/6837 using probe arrays or probe...C12Q 2525/185 incorporating bases where t...C12Q 2525/301 Hairpin oligonucleotidesC12Q 2561/101 TaqmanC12Q 2563/107 fluorescenceC12Q 2565/1015 labels being on the same ol...C12Q 2565/107 Alteration in the property ...C12Q 2565/519 characterised by the captur...
